Int J Stem Cells.  2023 May;16(2):123-134. 10.15283/ijsc22141.

The Calcineurin-Drp1-Mediated Mitochondrial Fragmentation is Aligned with the Differentiation of c-Kit Cardiac Progenitor Cells

• Rahman A
• Li Y
• Ismail NI
• Chan TK
• Li Y
• Xu D
• Zhou H
• Ong SB

• KMID: 2542826
• DOI: http://doi.org/10.15283/ijsc22141

Abstract

Objective
The heart contains a pool of c-kit⁺ progenitor cells which is believed to be able to regenerate. The differentiation of these progenitor cells is reliant on different physiological cues. Unraveling the underlying signals to direct differentiation of progenitor cells will be beneficial in controlling progenitor cell fate. In this regard, the role of the mitochondria in mediating cardiac progenitor cell fate remains unclear. Specifically, the association between changes in mitochondrial morphology with the differentiation status of c-kit⁺ CPCs remains elusive. In this study, we investigated the relationship between mitochondrial morphology and the differentiation status of c-kit⁺ progenitor cells.
Methods and Results
c-kit⁺ CPCs were isolated from 2-month-old male wild-type FVB mice. To activate differentiation, CPCs were incubated in α-minimal essential medium containing 10 nM dexamethasone for up to 7 days. To inhibit Drp1-mediated mitochondrial fragmentation, either 10 μM or 50 μM mdivi-1 was administered once at Day 0 and again at Day 2 of differentiation. To inhibit calcineurin, either 1 μM or 5 μM ciclosporin-A (CsA) was administered once at Day 0 and again at Day 2 of differentiation. Dexamethasone-induced differentiation of c-kit⁺ progenitor cells is aligned with fragmentation of the mitochondria via a calcineurin-Drp1 pathway. Pharmacologically inhibiting mitochondrial fragmentation retains the undifferentiated state of the c-kit+ progenitor cells.
Conclusions
The findings from this study provide an alternative view of the role of mitochondrial fusion-fission in the differentiation of cardiac progenitor cells and the potential of pharmacologically manipulating the mitochondria to direct progenitor cell fate.

Keyword

c-kit; Cardiac progenitor cells; Stem cell differentiation; Mitochondrial fragmentation; Drp1; Calcineurin

Figure

• 

  Fig. 1 Differentiation of c-kit+ CPCs is mediated by dexamethasone incubation. Representative epifluorescence images and quantification of (A) c-kit and (B) GATA-4 in the c-kit+ CPCs at Day 0 and 7-days post-dexamethasone treatment (n=80 cells per group; *p<0.05). (C) Representative Western blot of mitochondrial respiratory subunits in the c-kit+ CPCs at Day 0 and 7-days post-dexamethasone treatment.

• 

  Fig. 2 Differentiation of c-kit+ CPCs promotes changes in mitochondrial morphology. Dexamethasone incubation for 7 days causes (A) fragmentation of the mitochondria in the c-kit+ CPCs, (B) an increase in mitochondrial number, and (C) a reduction in the average size of the individual mitochondrion (n=80 cells per group; *p<0.05). (D) Mitochondrial fragmentation occurred as early as Day 2 following dexamethasone treatment (n=80 cells per group; *p<0.05).

• 

  Fig. 3 Mitochondrial fragmentation during c-kit differentiation is mediated by calcineurin-Drp1. (A) Representative Western blot images of mitochondrial-shaping proteins in c-kit+ CPCs at Day 0 and Day 7 following dexamethasone treatment. (B) Representative Western blot image of mitochondrial-shaping proteins following a time course of 7 days dexamethasone treatment. (C) Representative Western blot image profiling the changes in phosphorylated Drp1 at the S637 site following a time course of 7 days dexamethasone treatment. (D) Quantification of calcineurin activity during dexamethasone-differentiation of c-kit+ CPCs (n=3, *p<0.05 when compared with D0; †p<0.05 when compared with D7).

• 

  Fig. 4 Drp1 inhibition impairs mitochondrial fragmentation and c-kit differentiation. (A) Representative epifluorescence images of c-kit+ CPCs stained with antibodies against CoxIV (red), GATA-4 (green) with Hoescht (blue). (B) Quantification of c-kit+ CPCs with fragmented mitochondria following mdivi-1 treatment (n=80; *p<0.05 when compared with D0; †p<0.05 when compared with D7 with 50 μM mdivi-1). (C) GATA-4-positive c-kit+ CPCs following 7 days dexamethasone treatment in the presence of mdivi-1 (n=80; *p<0.05 when compared with D0; †p<0.05 when compared with D7 with 50 μM mdivi-1).

• 

  Fig. 5 Inhibition of calcineurin impairs mitochondrial fragmentation and differentiation of c-kit CPCs. (A) Representative epifluorescence images of c-kit+ CPCs stained with antibodies against CoxIV (red), GATA-4 (green) with Hoescht (blue). (B) Quantification of c-kit+ CPCs with fragmented mitochondria following CsA treatment (n=80; *p<0.05 when compared with D0; †p<0.05 when compared with D7 with 5 μM CsA). (C) GATA-4-positive c-kit+ CPCs following 7 days dexamethasone treatment in the presence of CsA (n=80; *p<0.05 when compared with D0; †p<0.05 when compared with D7 with 5 μM CsA).

Reference

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